The objective of the proposed research is to elucidate the structure-function relationship and the regulation of mammalian aminoacyl-tRNA synthetases. Besides aminoacylating cognate tRNAs for protein biosynthesis, mammalian synthetases participate in intracellular proteolysis, synthesis of signal nucleotides (AppppA and analogs) amino acid transport, autoimmune diseases and aging. At least eight of the mammalian aminoacyl-tRNA synthetases associate as a multi-enzyme complex. The synthetase complex presents a unique and interesting biochemical arrangement in terms of both structure and regulation. This research project proposes to delineate the fundamental principles in the high order of structural organization of proteins, and the evolved functions of such protein structure. The specific aims of the proposed projects are: 1) to clone and sequence mammalian aminoacyl-tRNA synthetases, and to obtain high level of expression of these synthetases in mammalian cells; 2) to determine the amino acid sequences involved in the assembly of the synthetase complex, and to study in vivo the roles of the synthetase complex in the synthetase turnover and the signal nucleotide biosynthesis; 3) to study in vitro the interaction of aminoacyl-tRNA synthetases with the synthetase complex, and to analyze its roles in the synthesis of signal nucleotide and mammalian protein biosynthesis; 4) to clone and map the genes of aminoacyl-tRNA synthetases, and determine the splicing sites and the regulatory sequences in the upstream flanking region. The methods to be used will be enzymological, structural and genetic. In view of the central role of proteins in all cell functions, understanding of the structure, function and the regulation of these essential enzymes in protein biosynthesis is of great importance.